In vivo screening method of therapeutic agent for memory/learning dysfunctions by schizophrenia

ABSTRACT

A method of evaluating memory/learning functions with the use of a model with glutamic acid N-methyl-D-aspartate (NMDA) type receptor hypofunction as an animal model for schizophrenia and with the use of reference memory task, wherein there has been found concrete means for detecting any differences in activity between typical anti-psychosis drugs and atypical anti-psychosis drugs is found. An in vivo animal model for screening of a therapeutic agent for improving cognitive dysfunction by schizophrenia is provided.

TECHNICAL FIELD

The present invention relates to an in vivo screening method of atherapeutic agent for improving memory/learning dysfunctions byschizophrenia.

BACKGROUND ART

Glutamic acid is a most popular excitatory neurotransmitter in thecentral nervous system, and the receptors thereof are classified broadlyinto an NMDA type, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic(AMPA) type, a kainate type, and a metabotropic type. It is revealedthat the NMDA type receptor plays an important role in the completion ofthe long-term potentiation (LTP), which is an electrophysiologicallybasal process of the memory/learning functions (cf., Science 285:1870-1874 (1999)). At the animal level, it is known that an NMDAreceptor antagonist may induce memory/learning dysfunctions in variousmemory/learning tasks such as a passive avoidance response, a radialmaze, a T or Y maze, a water maze, a place or object recognition, anautoshaping learning task, and a lever-pressing task (cf., Brain Res Rev41: 268-287 (2003)). It is also reported that PCP or ketamine, which isalso an NMDA receptor antagonist, induces cognitive dysfunctions inhumans (cf., Psychopharmacology 169: 215-233 (2003)). Namely, it is acommon opinion among the electrophysiological level, the animal leveland the human level that the NMDA type receptor plays an important rulein the memory/learning process.

It is the most widely-accepted hypothesis that the hypofunction of theNMDA type receptor is regarded as a mechanism of development ofschizophrenia. This hypothesis is established based on the followingfour points:

(i) PCP and ketamine, which are an NMDA receptor antagonist, induce themajor symptoms of schizophrenia in normal humans including cognitivedysfunction, positive-negative symptoms, and induce the exacerbation ofsymptoms in patients with schizophrenia (cf., Am J Psychiatry 158:1367-1377 (2001), Psychopharmacology 169: 215-233 (2003)):

(ii) There are clinical reports that glycine, D-serine andD-cycloserine, which have been known to elevate NMDA receptor functions,enhance the drug efficacy of anti-psychotic drugs in patients withschizophrenia, and improve negative symptoms and cognitive dysfunctions(cf., Am J Psychiatry 158: 1367-1377 (2001), Psycho-pharmacology 169:215-233 (2003)):

(iii) The variation in the amount of glutamic acid per se, or the amountof the endogenous substance: N-acetyl-aspartyl glutamate (NAAG) havingan NMDA antagonistic activity is observed in patients with schizophrenia(cf., Am J Psychiatry 158: 1367-1377 (2001)), and the change in theamount of mRNA and proteins of NMDA type receptor subunit and NMDAreceptor-related protein is observed in patients with schizophrenia(cf., Am J Psychiatry 157: 1811-1823 (2000), Am J Psychiatry 160:1100-1109 (2003), Society for Neuroscience Program No. 754.4. (2003)):

(iv) A series of genes which were found as a gene relating to the onsetof schizophrenia (Neuregulin 1, G72, dysbindin, calcineurin) are genesbeing capable of modifying NMDA receptor functions (cf., Proc Natl AcadSci USA 99: 13365-13367 (2002), Neuron 40: 881-884 (2003)). From theabove facts, it is generally considered that the dysfunctions induced byNMDA type receptor antagonists are models to reflect the dysfunctions byschizophrenia.

Schizophrenia is associated with various cognitive dysfunctions such asattention, memory, learning, executive functions, but it is reportedthat among these functions, especially a certain memory function isselectively and seriously damaged. Namely, memory is classified broadlyinto procedural memory and declarative memory. The declarative memory isfurther classified into short-term memory/working memory and a long-termmemory/reference memory. It is reported that in schizophrenia, thedeclarative memory including both of the working memory and thereference memory is selectively damaged, and further, among them, thedamage of the reference memory is most serious. Recently, it is reportedthat the cognitive dysfunction including such a reference memorydysfunction is the most important predictive factor of social dailyability and professional ability, and a quality of life of patients withschizophrenia. Then, at the moment, the cognitive dysfunction ispositioned as a core symptom of schizophrenia (cf., Psychopharmacology169: 213-214 (2003), Proc Natl Acad Sci USA 96: 13591-13593 (2002),Psychoneuroendocrinology 28: 27-38 (2003), Psychiat Clin N Am 26: 25-40(2003)). Under these circumstances, many clinical trials are being donewith respect to the effects of the existing anti-psychotic agents onvarious cognitive dysfunctions by schizophrenia (cf., Psychopharmacology162: 11-17 (2002), Psychoneuroendocrinology 28: 27-38 (2003), PsychiatClin N Am 26: 25-40 (2003)), and there is being submitted an evidencethat a typical anti-psychotic agent, haloperidol, is ineffective, whilesome of atypical anti-psychotic agents are effective (cf., J. ClinPsychiatry 65: 361-372, Psychoneuroendocrinology 28: 27-38 (2003),Psychiat Clin N Am 26: 25-40 (2003)). However, the drug efficacy ofthese existing drugs are not sufficient enough, and hence, it has beendiscussed that it is important to develop a therapeutic agent forcognitive dysfunctions by schizophrenia (cf., Science 299: 350-351(2003)).

On the other hand, in the research and development of therapeutic agentsfor human diseases, it is generally essential to develop an animal modelbeing suitable for screening thereof. In such an animal model, the facevalidity (similarities of symptoms), the construct validity(similarities of the mechanism of development of symptoms), and thepredictive validity (predictability of clinical drug efficacy), andfurther, the easiness being suitable for screening are required.However, it is considered that an animal model for cognitivedysfunctions by schizophrenia satisfying such requirements is quitelimited at the moment. Namely, as an animal model for schizophreniabeing capable of satisfying the above-mentioned requirements,PCP-induced models showing prepulse inhibition or social interactionfailure can be exemplified (cf., Prog Neuropsychopharmacol BiolPsychiaty 27: 1071-1079 (2003)). It is known that the drug efficacy ofatypical anti-psychotic agents, but that of a typical anti-psychoticagent can be selectively detected in these models, and the resultsobtained in these animal models partially reflect the clinical effectsof a drug on schizophrenia. However, it is considered that among theseanimal models the former one may reflect the disorder of thesensorimotor gating function in schizophrenia, while the latter mayreflect the negative symptoms of schizophrenia such a social withdrawal.Thus, as mentioned above, an easy model for evaluating drug efficacy,(1) being capable of reflecting reference memory dysfunction, which isthe most serious cognitive dysfunction in schizophrenia (i.e., facevalidity, similarities of symptoms); (2) being associated with NMDAreceptor hypofunction, which is a most possible cause for schizophrenia(i.e., construct validity, similarities of the mechanism of onset); and(3) being capable of detecting an excellent drug efficacy of an atypicalanti-psychotic agent rather than that of a typical anti-psychotic agent(predictive validity, predictability of clinical drug efficacy), isconsidered to be quite useful in the research and development of atherapeutic agent for cognitive dysfunction, a core symptom ofschizophrenia, but such an animal model has not been known yet untilnow.

In animals, various tasks consisting of both of the training session,and the testing session being carried out after a prescribed period fromthe testing session, can be used in order to study a reference memory.In the training session, the animals are made to learn an avoidanceresponse such as electroconvulsive shock (passive or active avoidanceresponse), a task of reaching to a platform which is not visible belowwater (a water maze task), a task of fetching a food after gettingthrough a maze task or a task of avoiding an electroconvulsive shock(radial maze task, Y or T maze task), a task of recognizing andsearching a novel place or object, a task of pressing a lever forobtaining a food, etc., and further they are made to acquire the memorythereof. The animals are returned to exactly the same experimentalenvironment after a prescribed period therefrom, and they are tested ifthey can retrieve the acquired memory. In the reference memory tasks,the animals can memorize all of the specific environment and stimuluswhich are given to them in a specific order in the training session. Inorder to correctly evaluate at the testing session the success andfailure of the memory acquisition in the training session, it isnecessary to carry out the training session and the learning sessionunder exactly the same environment and stimulus. Especially, there is areport that animals show an abnormal memory retrieval when ethanol or anNMDA type receptor antagonist is administered only in the trainingsession of the reference memory tasks, but not in the testing session(cf., Brain Res 706: 227-232 (1996), Pharmcol Biochem Behav 69: 585-593(2001)). In such cases, the animals acquire a reference memory dependingon the environment in the brain, which is induced by the administrationof a drug, and in fact, it is proved that by administering a drug bothin the training session and the testing session, the acquired memory cancorrectly be retrieved. Such a phenomenon is usually calledstate-dependency. In the evaluation of the reference memory functions,it is sometimes necessary to give sufficient consideration to thestate-dependency in some cases, and it is necessary to avoid anyartificial misjudgment on the evaluation of memory functions due tostate-dependency. As mentioned above, in order to avoid anystate-dependency effect by an agent for inducing memory/learningdysfunctions such as an NMDA type receptor antagonist, and to evaluatethe reference memory/learning function, there is a method comprisingadministering an agent for inducing memory/learning dysfunctions in bothof the training session and the testing session. Further, as analternative method, it is easily speculated to utilize a methodcomprising chronically administering an agent for inducingmemory/learning dysfunctions during the period including the trainingsession and the testing session. In addition, instead of administeringan agent for inducing memory/learning dysfunctions, it may be possibleto utilize a method comprising expressing a chronic dysfunction duringthe period including the training session and the testing sessions ofthe reference memory tasks with the use of a gene engineering technique.For example, an NMDA type receptor subtype NR1 knockdown or NR2A subtypeknockout animal are already produced as a concrete example for an animalmodel showing a chronic hypofunction of NMDA type receptor (cf., Cell98: 427-436 (1999), J Neurosci 21: 750-757 (2001)).

DISCLOSURE OF INVENTION

The present invention provides a screening method of a therapeutic agentfor memory/learning dysfunctions by schizophrenia. More particularly,the present invention provides an animal model for reference memorydysfunction caused by hypofunction of NMDA type receptor as a simpleanimal model for schizophrenia providing the predictability of theclinical drug efficacy of the existing therapeutic agents.

The present inventors have intensively studied in order to solve theabove problems, and found that the reference memory dysfunction in theanimals where NMDA type receptor hypofunction is induced both in thetraining session and the testing session is specifically improved by anatypical anti-psychotic agent but not by a typical anti-psychotic agent,and further they have confirmed that this evaluation system is a verysimple and highly-reproducible evaluation system, and finally they haveaccomplished the present invention.

Namely, the present invention relates to the following features:

[1] An in vivo screening method for predicting whether or not a testcompound is capable of improving the memory/learning dysfunctions byschizophrenia, wherein said method comprises a step of evaluating thememory/learning functions by employing a model showing glutamic acidN-methyl-D-aspartate (NMDA) type receptor hypofunction as an animalmodel for schizophrenia, and a reference memory task.

[2] The method according to the above [1], wherein the reference memorytask is a passive avoidance task, an active avoidance task, a water mazetask, a radial maze task, a T or Y maze task, a place recognition task,an object recognition task, an autoshaping learning task, or alever-pressing task.

[3] The method according to the above [1], wherein the reference memorytask is composed of two sessions of training and testing, and in thetraining session the animals are made to learn either of the tasksdescribed in the above [2] and to acquire the memory of said task, andin the testing session being carried out after a prescribed period fromthe training session, the retention and retrieval ability of said memoryof the animals are quantified.

[4] The method according to the above [1], wherein the model showing anNMDA type receptor hypofunction is produced by administering a compoundhaving an NMDA type receptor antagonistic activity, e.g., MK-801,phencyclidine (PCP), ketamine, or a derivative thereof, to the animalsin both of the training session and the testing session of the referencememory task, or by chronically administering said compound or aderivative thereof to the animals during the period including thetraining session and the testing session.

[5] The method according to the above [1], wherein the model showing anNMDA type receptor hypofunction is an animal model associated with anNMDA type receptor hypofunction due to variation, overexpression, ordeficiency of gene of constitutive proteins or relevant proteins of anNMDA type receptor in both of the training session and the testingsession of the reference memory task.

[6] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises a substance selected by a screeningmethod as set forth in any one of the above [1] to [5] as an activeingredient.

[7] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises a serotonin 5-HT 1A antagonist selectedby a screening method as set forth in any one of the above [1] to [5] asan active ingredient.

[8] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises a choline acetylase inhibitor selected bya screening method as set forth in any one of the above [1] to [5] as anactive ingredient.

[9] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises aricept as an active ingredient.

[10] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises quetiapine as an active ingredient.

[11] The therapeutic agent for the memory/learning dysfunctions byschizophrenia according to the above [10], which comprises as an activeingredient quetiapine in a daily dose of 5 to 270 mg.

[12] The therapeutic agent for the memory/learning dysfunctions byschizophrenia according to the above [10], which comprises as an activeingredient quetiapine in a daily dose of 15 to 90 mg.

[13] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises clozapine as an active ingredient.

[14] The therapeutic agent for the memory/learning dysfunctions byschizophrenia according to the above [13], which comprises as an activeingredient clozapine in a daily dose of 0.2 to 34.5 mg.

[15] The therapeutic agent for the memory/learning dysfunctions byschizophrenia according to the above [13], which comprises as an activeingredient clozapine in a daily dose of 0.7 to 11.5 mg.

[16] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises as an active ingredient an imidederivative of the formula [1]:

{wherein Z is a group of the formula:

(in which B is a carbonyl or a sulfonyl; R¹ R², R³ and R⁴ areindependently a hydrogen atom or a lower alkyl, provided that R¹ and R²,or R¹ and R³ may combine each other to form a hydrocarbon ring, or R¹and R³ may combine each other to form an aromatic hydrocarbon ring; saidhydrocarbon ring may optionally be cross-linked with a lower alkylene oran oxygen atom; said lower alkylene and hydrocarbon ring may optionallybe substituted by at least one alkyl; and n is 0 or 1),D is a group of the formula:

(in which A is a hydrocarbon ring optionally be cross-linked with alower alkylene or an oxygen atom; said lower alkylene and saidhydrocarbon ring may optionally be substituted by at least one alkyl;and p and q are independently 0, 1 or 2),G is N, CH or COH, and —Ar is an aromatic heterocyclic group, anaromatic hydrocarbon group, benzoyl, phenoxy, or phenylthio, orG is a carbon atom, and —Ar is a biphenylmethylidene,where said aromatic heterocyclic group, aromatic hydrocarbon group,benzoyl, phenoxy, phenylthio, and biphenylmethylidene may optionally besubstituted by at least one group selected from a lower alkyl, a loweralkoxy and a halogen atom},or an acid addition salt thereof.[17] The therapeutic agent for the memory/learning dysfunctions byschizophrenia comprising as an active ingredient the imide derivative oran acid addition salt thereof according to the above [16], wherein Ar isan aromatic heterobicyclic group, naphthyl, benzoyl, phenoxy orphenylthio, and G is N, CH or COH, or —Ar is a biphenylmethylidene, andG is a carbon atom (said aromatic heterobicyclic group, naphthyl,benzoyl, phenoxy, phenylthio and biphenylmethylidene may optionally besubstituted by at least one group selected from a lower alkyl, a loweralkoxy and a halogen atom).[18] The therapeutic agent for the memory/learning dysfunctions byschizophrenia comprising as an active ingredient the imide derivative oran acid addition salt thereof according to the above [16], wherein Ar isan aromatic heterocyclic group condensed with a benzene ring, ornaphthyl, benzoyl, phenoxy or phenylthio (said aromatic heterocyclicgroup condensed with a benzene ring, naphthyl, benzoyl, phenoxy, andphenylthio may optionally be substituted by at least one group selectedfrom a lower alkyl, a lower alkoxy and a halogen atom), and G is N, CHor COH.[19] The therapeutic agent for the memory/learning dysfunctions byschizophrenia comprising as an active ingredient the imide derivative oran acid addition salt thereof according to the above [16], wherein Z isa group of the formula:

(in which -L- is a single bond or a double bond, E is a lower alkyleneoptionally substituted by a lower alkyl, or an oxygen atom, R⁵ is ahydrogen atom or a lower alkyl, and B is the same as defined in theabove [14]);a group of the formula:

(in which -L-, E, R⁵ and B are as defined above);a group of the formula:

(in which R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ are independentlya hydrogen atom or a lower alkyl, or the adjacent two groups of R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ may combine each other to form adouble bond, and B is as defined above);a group of the formula:

(in which R¹⁶ and R¹⁷ are independently a hydrogen atom or a loweralkyl, or R¹⁶ and R¹⁷ may combine each other to form a saturatedhydrocarbon ring, and R⁵ and B are as defined above); ora group of the formula:

(in which B is as defined above).[20] A therapeutic agent for the memory/learning dysfunctions byschizophrenia comprising as an active ingredient the imide derivative oran acid addition salt thereof, wherein the compound of the formula [1]is lurasidone:

[21] A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises as an active ingredient a compound of theformula (2):

wherein Z is a divalent sulfur, imino, or lower alkylimino;R₁₁ is a hydrogen atom or an alkyl having 1 to 5 carbon atoms;R₁₂ is a hydrogen atom, an alkyl having 1 to 5 carbon atoms, a phenyl,an R₁₅-substituted phenyl, an aminoalkyl having 1 to 5 carbon atoms, alower alkylaminoalkyl having 2 to 8 carbon atoms, a lower alkylamino, anamino, or a lower alkylamino; orR₁₁ and R₁₂ may combine each other together with N to form a1-pyrrolidinyl, piperidino, morpholino, thiomorpholino, 1-piperazinyl, a4-lower alkyl-1-piperazinyl, a 4-(hydroxy-lower alkyl)-1-piperazinyl ora 4-(lower alkoxy-lower alkyl)-1-piperazinyl; andR₁₃, R₁₄, and R₁₅ are independently a hydrogen atom, a halogen atom, ahydroxy group, a trifluoromethyl, a lower alkyl, a lower alkoxy, or alower alkylthio,or an acid addition salt thereof.

The present invention provides a concrete method for evaluating amemory/learning improving activity being specific to certain atypicalanti-psychotic agents but not to a typical anti-psychotic agenthaloperidol. The results obtained by this method are in agreement withthe clinical findings that haloperidol shows no improving activity ofcognitive dysfunctions, and that atypical anti-psychotic agents exhibitan improving activity of cognitive dysfunctions. As a result, it becomespossible to provide a screening method of a therapeutic agent forcognitive dysfunctions by schizophrenia, and further provides a concretetherapeutic agent therefor. Actually, by the present method, thememory/learning improving efficacy is recognized with respect tolurasidone, which is under development as a candidate for a noveltherapeutic agent for schizophrenia, noradrenaline α2 receptorantagonist 1-(2-pyrimidyl)piperazine dihydrochloride (hereinafter,referred to as 1-PP), and serotonin 5-HT 1A receptor antagonist inaddition to risperidone, clozapine or quetiapine, by which a candidatefor a novel agent for improving cognitive dysfunctions by schizophreniacan be provided. In addition, the memory/learning improving activity ofclozapine and quetiapine is observed at a dose by 10 times or more lowerthan the dose at which they exhibit an anti-psychotic activity, andhence, a different action mechanism can be speculated. Further, since itis suggested that the retrieval of reference memory task being acquiredwith improvement by a drug is done with depending on the state of theNMDA type receptor hypofunction (state-dependent), as a novel andconcrete method for evaluating the reference memory dysfunctionimproving activity of a drug, a method where the NMDA type receptorhypofunction is induced during the period including the training sessionand the testing session is effective.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention relates to an in vivo screening method forpredicting whether or not a test compound is capable of improving thememory/learning dysfunctions by schizophrenia, wherein said methodcomprises a step of evaluating the memory/learning function by employinga model showing an NMDA type receptor hypofunction as an animal modelfor schizophrenia and a reference memory task.

The reference memory task includes, for example, a passive avoidancetask, an active avoidance task, a water maze task, a radial maze task, aT or Y maze task, a place recognition task, an object recognition task,an autoshaping learning task, and a lever-pressing task.

The reference memory task is composed of two sessions such as trainingand testing. In the training session, an animal is made to learn acertain task and further made to acquire a memory thereof. After aprescribed period from the learning session, the testing session iscarried out, and the retention and retrieval of the memory of task atthe testing session is quantified.

A compound having an NMDA type receptor antagonistic activity, e.g.,MK-801, PCP, ketamine, or a derivative thereof, is administered toanimals during both in the training session and the testing session ofthe reference memory task, or chronically administered to animals duringthe period including the training session and the testing session, andthese animals are used as an animal model showing an NMDA type receptorhypofunction.

Alternatively, an animal model associated with an NMDA type receptorhypofunction due to variation, overexpression or deficiency of gene ofconstitutive proteins or relevant proteins of NMDA type receptor in bothof the training session and the testing session in the reference memorytask can be used as a model showing an NMDA type receptor hypofunction.

Hereinafter, the present invention is illustrated in more detail byExamples, but the present invention should not be construed to belimited thereto.

EXAMPLE 1

(Method)

Wistar male rats (7 weeks old) were used. Haloperidol (a typicalanti-psychotic agent), clozapine, quetiapine, risperidon, olanzapine oraripiprazole (atypical anti-psychotic agent), or lurasidone being underdevelopment as a novel anti-psychotic agent was suspended in a 0.5%methyl cellulose (MC) and the resultant suspension was used as a testcompound. Serotonin 5-HT 1A receptor antagonist WAY-100635 ornoradrenaline α2 receptor antagonist 1-PP was dissolved in aphysiological saline solution (Otsuka Pharmaceutical Co., Ltd.) and usedas a test compound. As an NMDA type receptor antagonist, MK-801 hydrogenmaleate (SIGMA-ALDRICH M-107) was dissolved in a physiological salinesolution (Otsuka Pharmaceutical Co., Ltd.). A test compound (0.3 to 10mg/kg) or a 0.5% MC or a physiological saline solution as a control wasorally or interperitoneally administered to the animals one hour priorto the training session of the passive avoidance task, and MK-801 (0.05mg/kg) or a physiological saline solution as a control wassubcutaneously administered 30 minutes prior to both of the trainingsession and the testing session. For the evaluation of state-dependencyof MC-801, MK-801 was administered before the training session, and aphysiological saline solution was administered before the testingsession instead. The dosing value thereof was 5 ml/kg for each.

The step-through type passive avoidance response test was carried out inthe following manners with the use of an apparatus consisting of alight-dark box, a slide door dividing the light-dark box and a shockgenerator (manufactured by O'hara & Co., Ltd., PA-2030A, PA-3001A) as anexperimental apparatus. Namely, on Day 1 of the experiment, after a testcompound and MK-801 were administered, the rats were put into the lightbox of the experimental apparatus where the back of each rat wasdirected to the dark box. Then, 10 seconds later, a slide door set atthe border between the dark box and the light box was opened. Due to thehabits of the rats, once the rats entered into the dark box, the slidedoor was quickly closed. At three seconds after entering into the darkbox, an electroconvulsive shock (0.5 mA, for 3 seconds) was given to therats. The period between the time just after the slide door was openedand the time at which the rats entered into the dark box was measured asa step-through latency. As to the animals which did not enter into thedark room even after 300 seconds, the training was terminated, and thoseanimals were dropped in the following experiment for the reasons oftraining failure. On Day 2 of the experiment, the testing session wascarried out about 24 hours after the training session. Thirty minutesprior to the testing session, MK-801 or a vehicle thereof, i.e., aphysiological saline solution, was subcutaneously administered to therats. The procedures of the testing session were carried out in the samemanner to the training session except that no electroconvulsive shockwas given. The step-through latency at the testing session was measuredup to 300 seconds, and the step-through latency over 300 seconds wasregarded as 300 seconds. The number of animals which showed 300 secondsat the testing session was counted, and the ratio thereof was calculatedin percentages (as defined as a % of animals avoiding) in each group.The statistical analysis of the % of animals avoiding was done by x²test with Bonferroni's correction. The animals were used in a group of10 to 25 animals per group, and the data was expressed by percentage.

(Results)

First, a drug was administered alone without MK-801, and a dose of thedrug to induce the memory/learning dysfunctions in the passive avoidanceresponse was determined. Further a dose less than that dose and toinduce no memory/learning dysfunctions was used as an administrationdose in the MK/801 models (Table 1).

The animals to which MK-801 was administered before the trainingsession, and a physiological saline solution was administered before thetesting session showed the nearly equal decrease in the step-throughlatency to that of the animals to which MK-801 was administered both inthe training session and the learning session. That is, it was foundthat the memory dysfunction observed in the cases where MK-801 wasadministered only before the training session, or before both of thetraining session and the testing sessions, is not a memory retrievaldysfunction but memory acquisition dysfunction.

Next, the effects of drugs on the memory dysfunction observed in thecases where MK-801 was administered in both of the training session andthe testing session were studied. In the group to which MK-801 was notadministered, the animals showing 300 seconds of step-through latency (%of animals avoiding) was 75 to 80% in all of the experiments. On thecontrary, the % of animals avoiding became 0 to 5% in all of theexperiments by the administration of MK-801. The effects of the testcompounds on the % of animals avoiding were shown in Table 1. As isshown in Table 1, there were differences of the drug efficacy strengthamong individual drugs. That is, while no drug efficacy was observedwith respect to either a typical anti-psychotic agent haloperidol, oratypical anti-psychotic agents olanzapine, aripiprazole, but asignificant improving activity was observed with respect to atypicalanti-psychotic agents clozapine, quetiapine, lurasidone, and serotonin5-HT 1A receptor antagonist WAY-100635, noradrenalin α2 receptorantagonist 1-PP.

Interestingly, it is known that a dose to inhibit 50% ofmethamphetamine-induced hyperponesis in rats (ED50, mg/kg, p.o.), whichreflects anti-psychotic activity, is 65 and about 100 with respect toclozapine and quetiapine, respectively. That is, it is suggested thatclozapine and quietiapine show a memory/learning dysfunction improvingactivity at a dose 65-272 times or 10 times lower than the dose thereofshowing anti-psychotic activity, ED50, respectively. Thus, it becameapparent that these drugs show its memory/learning dysfunction improvingactivity in a different action mechanism from that of the anti-psychoticactivity thereof. Since the clinical daily dose of clozapine as ananti-psychotic agent is 150-750 mg, then a dose thereof for improvingmemory/learning dysfunctions by schizophrenia should be 0.2-34.5 mg,preferably 0.7-11.5 mg, which are obtained by calculating 150/217-750/65(i.e., 0.7-11.5 mg) and broadening the obtained range into eitherdirection by about 3 times. On the other hand, since the clinical dailydose of quetiapine as an anti-psychotic agent is 150-900 mg, then a dosethereof for improving memory/learning dysfunctions by schizophreniashould be 5-270 mg, preferably 15-90 mg, which is obtained bycalculating 150/10-900/10 (i.e., 15-90 mg) and broadening the obtainedrange into either direction by about 3 times.

Finally, with respect to lurasidone and 1-PP, which showed the mostremarkable memory/learning dysfunction improving activity, when theeffects thereof on the memory dysfunction in cases where MK-801 wasadministered only in the training session were studied, either of thesedrugs showed no drug efficacy. From these results, it was proved thatthe memory of passive avoidance response being acquired with improvementby lurasidone and 1-PP depends on the state of MK-801 administration,i.e., state-dependent.

EXAMPLE 2

In the procedures of Example 1, PCP HCl (0.75 mg/kg) was subcutaneouslyadministered instead of MK-801 (0.05 mg/kg) to animals prior to both ofthe training session and the testing session of the memory/learning taskto induce memory/learning dysfunctions, and the memory/learningdysfunction improving activity of a test compound can be evaluated.

EXAMPLE 3

The memory/learning dysfunction improving activity of a test compoundcan be evaluated under the exactly same conditions as those in Examples1 and 2, except that ketamine is used instead of MK-801 or PCP HCl.

EXAMPLE 4

In the procedures of Examples 1 to 3, instead of subcutaneousadministration of an NMDA type receptor antagonist such as MK-801, PCPHCl or ketamine prior to both of the training session and the testingsession, these drugs are injected to an ALZET osmotic pump and implantedunder the skin of the back side of the animals so that these drugs arechronically administered under the skin during the period including thetraining session and the testing session, and a memory/learningdysfunctions can be induced thereby, and then the memory/learningdysfunction improving activity of a test compound thereon can beevaluated.

EXAMPLE 5

Instead of the method of using an NMDA type receptor antagonist as shownin the above Examples, an animal showing a variation, overexpression ordeficiency of gene of constitutive proteins or relevant proteins of theNMDA type receptor can be utilized.

EXAMPLE 6

In the procedures of Examples 1 to 5, either one of an active avoidancetask, a water maze task, a radial maze task, a T or Y maze task, a placerecognition task, an object recognition task, an autoshaping learningtask, and a lever-pressing task can be employed instead of a passiveavoidance task, and the exactly same experiment as Example 1 can becarried out.

Table 1 shows the effects of the drugs on MK-801-induced memory/learningdysfunctions in the passive avoidance task in rats.

To be more precise, Table 1 shows the effects of the drugs in terms ofthe percentage of the animals acquiring passive avoidance response(i.e., the percentage of the animals showing a step-through latency of300 seconds or more in the testing session, that is, the percentage ofthe animals showing a remarkable memory retention, a % of animalsavoiding) in the passive avoidance response test in the rats to whichMK-801, an NMDA type receptor antagonist, was administered prior to bothof the training session and the testing session.

In a similar passive avoidance response test, a minimum dose of a drugto induce memory/learning dysfunctions by a single application thereofis shown in the right edge column of Table.

The figures in Table mean a % of animals avoiding. In a similar passiveavoidance response test, a minimum dose of a drug to inducememory/learning dysfunctions by a single application thereof is shown inthe right edge column of Table. TABLE 1 Group Minimum dose to treatedwith Groups treated induce memory MK-801 with MK-801 + dysfunction by(0.05 mg/kg, Drug (mg/kg, p.o.) single application, Drug Vehicle groups.c.) 0.1 0.3 1 3 10 mg/kg, p.o (MK-801 administered prior to trainingsession) Lurasidone 80 0 — 20 20 30 20 >30 1-PP 92 0 — — —  8 — —(MK-801 administered prior to both of training and testing sessions)Haloperidol 80 0  0  5 15 10 — 10 Olanzapine 80 0 — 15 20 20 10 3Risperidone 76 4 20 20 28 10 — 3 Clozapine 80 4 —  44*  36* 20 10 10Quetiapine 75 5 — 10 20 30  50* 30 Aripiprazole 80 4 — — 16 16 — 10Lurasidone 80 0 —  40*  35*  75*  70* >30 1-PP 80 13 — —  47*  67* — —WAY-1000635 75 5 — 30  40* 30 — —n = 10-25*P < 0.05 vs Group treated with MK-801 (0.05 mg/kg s.c.)

INDUSTRIAL APPLICABILITY

The present invention provides an in vivo screening method of atherapeutic agent for improving memory/learning dysfunctions byschizophrenia.

1. An in vivo screening method for predicting whether or not a testcompound is capable of improving the memory/learning dysfunctions byschizophrenia, wherein said method comprises a step of evaluating thememory/learning functions by employing a model showing glutamic acidN-methyl-D-aspartate (NMDA) type receptor hypofunction as an animalmodel for schizophrenia, and a reference memory task.
 2. The methodaccording to claim 1, wherein the reference memory task is a passiveavoidance task, an active avoidance task, a water maze task, a radialmaze task, a T or Y maze task, a place recognition task, an objectrecognition task, an autoshaping learning task, or a lever-pressingtask.
 3. The method according to claim 1, wherein the reference memorytask is composed of two sessions of training and testing, and in thetraining session the animals are made to learn either of the tasksdescribed in claim 2 and to acquire the memory of said task, and in thetesting session being carried out after a prescribed period from thetraining session, the retention and retrieval ability of said memory ofthe animals are quantified.
 4. The method according to claim 1, whereinthe model showing an NMDA type receptor hypofunction is produced byadministering a compound having an NMDA type receptor antagonisticactivity, e.g., MK-801, phencyclidine (PCP), ketamine, or a derivativethereof, to the animals in both of the training session and the testingsession of the reference memory task, or by chronically administeringsaid compound or a derivative thereof to the animals during the periodincluding the training session and the testing session.
 5. The methodaccording to claim 1, wherein the model showing an NMDA type receptorhypofunction is an animal model associated with an NMDA type receptorhypofunction due to variation, overexpression, or deficiency of gene ofconstitutive proteins or relevant proteins of an NMDA type receptor inboth of the training session and the testing session of the referencememory task.
 6. A therapeutic agent for the memory/learning dysfunctionsby schizophrenia, which comprises a substance selected by a screeningmethod as set forth in any one of claims 1 to 5 as an active ingredient.7. A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises a serotonin 5-HT 1A antagonist selectedby a screening method as set forth in any one of claims 1 to 5 as anactive ingredient.
 8. A therapeutic agent for the memory/learningdysfunctions by schizophrenia, which comprises a choline acetylaseinhibitor selected by a screening method as set forth in any one ofclaims 1 to 5 as an active ingredient.
 9. A therapeutic agent for thememory/learning dysfunctions by schizophrenia, which comprises ariceptas an active ingredient.
 10. A therapeutic agent for the memory/learningdysfunctions by schizophrenia, which comprises quetiapine as an activeingredient.
 11. The therapeutic agent for the memory/learningdysfunctions by schizophrenia according to claim 10, which comprises asan active ingredient quetiapine in a daily dose of 5 to 270 mg.
 12. Thetherapeutic agent for the memory/learning dysfunctions by schizophreniaaccording to claim 10, which comprises as an active ingredientquetiapine in a daily dose of 15 to 90 mg.
 13. A therapeutic agent forthe memory/learning dysfunctions by schizophrenia, which comprisesclozapine as an active ingredient.
 14. The therapeutic agent for thememory/learning dysfunctions by schizophrenia according to claim 13,which comprises as an active ingredient clozapine in a daily dose of 0.2to 34.5 mg.
 15. The therapeutic agent for the memory/learningdysfunctions by schizophrenia according to claim 13, which comprises asan active ingredient clozapine in a daily dose of 0.7 to 11.5 mg.
 16. Atherapeutic agent for the memory/learning dysfunctions by schizophrenia,which comprises as an active ingredient an imide derivative of theformula [1]:

{wherein Z is a group of the formula:

(in which B is a carbonyl or a sulfonyl; R¹ R², R³ and R⁴ areindependently a hydrogen atom or a lower alkyl, provided that R¹ and R²,or R¹ and R³ may combine each other to form a hydrocarbon ring, or R¹and R³ may combine each other to form an aromatic hydrocarbon ring; saidhydrocarbon ring may optionally be cross-linked with a lower alkylene oran oxygen atom; said lower alkylene and hydrocarbon ring may optionallybe substituted by at least one alkyl; and n is 0 or 1), D is a group ofthe formula:—(CH₂)_(p)-A-(CH₂)_(q)— (in which A is a hydrocarbon ring optionally becross-linked with a lower alkylene or an oxygen atom; said loweralkylene and said hydrocarbon ring may optionally be substituted by atleast one alkyl; and p and q are independently 0, 1 or 2), G is N, CH orCOH, and —Ar is an aromatic heterocyclic group, an aromatic hydrocarbongroup, benzoyl, phenoxy, or phenylthio, or G is a carbon atom, and —Aris a biphenylmethylidene, where said aromatic heterocyclic group,aromatic hydrocarbon group, benzoyl, phenoxy, phenylthio, andbiphenylmethylidene may optionally be substituted by at least one groupselected from a lower alkyl, a lower alkoxy and a halogen atom}, or anacid addition salt thereof.
 17. The therapeutic agent for thememory/learning dysfunctions by schizophrenia comprising as an activeingredient the imide derivative or an acid addition salt thereofaccording to claim 16, wherein Ar is an aromatic heterobicyclic group,naphthyl, benzoyl, phenoxy or phenylthio, and G is N, CH or COH, or —Aris a biphenylmethylidene, and G is a carbon atom (said aromaticheterobicyclic group, naphthyl, benzoyl, phenoxy, phenylthio andbiphenylmethylidene may optionally be substituted by at least one groupselected from a lower alkyl, a lower alkoxy and a halogen atom).
 18. Thetherapeutic agent for the memory/learning dysfunctions by schizophreniacomprising as an active ingredient the imide derivative or an acidaddition salt thereof according to claim 16, wherein Ar is an aromaticheterocyclic group condensed with a benzene ring, or naphthyl, benzoyl,phenoxy or phenylthio (said aromatic heterocyclic group condensed with abenzene ring, naphthyl, benzoyl, phenoxy, and phenylthio may optionallybe substituted by at least one group selected from a lower alkyl, alower alkoxy and a halogen atom), and G is N, CH or COH.
 19. Thetherapeutic agent for the memory/learning dysfunctions by schizophreniacomprising as an active ingredient the imide derivative or an acidaddition salt thereof according to claim 16, wherein Z is a group of theformula:

(in which -L- is a single bond or a double bond, E is a lower alkyleneoptionally substituted by a lower alkyl, or an oxygen atom, R⁵ is ahydrogen atom or a lower alkyl, and B is the same as defined in claim14); a group of the formula:

(in which -L-, E, R⁵ and B are as defined above); a group of theformula:

(in which R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ are independentlya hydrogen atom or a lower alkyl, or the adjacent two groups of R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ may combine each other to form adouble bond, and B is as defined above); a group of the formula:

(in which R¹⁶ and R¹⁷ are independently a hydrogen atom or a loweralkyl, or R¹⁶ and R¹⁷ may combine each other to form a saturatedhydrocarbon ring, and R⁵ and B are as defined above); or a group of theformula:

(in which B is as defined above).
 20. A therapeutic agent for thememory/learning dysfunctions by schizophrenia comprising as an activeingredient the imide derivative or an acid addition salt thereof,wherein the compound of the formula [1] is lurasidone:


21. A therapeutic agent for the memory/learning dysfunctions byschizophrenia, which comprises as an active ingredient a compound of theformula (2):

wherein Z is a divalent sulfur, imino, or lower alkylimino; R₁₁ is ahydrogen atom or an alkyl having 1 to 5 carbon atoms; R₁₂ is a hydrogenatom, an alkyl having 1 to 5 carbon atoms, a phenyl, an R₁₅-substitutedphenyl, an aminoalkyl having 1 to 5 carbon atoms, a loweralkylaminoalkyl having 2 to 8 carbon atoms, a lower alkylamino, anamino, or a lower alkylamino; or R₁₁ and R₁₂ may combine each othertogether with N to form a 1-pyrrolidinyl, piperidino, morpholino,thiomorpholino, 1-piperazinyl, a 4-lower alkyl-1-piperazinyl, a4-(hydroxy-lower alkyl)-1-piperazinyl or a 4-(lower alkoxy-loweralkyl)-1-piperazinyl; and R₁₃, R₁₄, and R₁₅ are independently a hydrogenatom, a halogen atom, a hydroxy group, a trifluoromethyl, a lower alkyl,a lower alkoxy, or a lower alkylthio, or an acid addition salt thereof.